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I have developed the HPLC method for quantify a pharmaceutical compound in plasma. Now I want to determinate the partition of my compound between plasma and cellular fraction.
1. After the spike in blood with a concentration of my compound, I have used two methods: a. I separate the plasma and the cellular fraction by centrifugation; I disrupted the cellular fraction; I precipitate the proteins and/or the cellular residuals by a suitable precipitating agent.
b. I divide the blood into two aliquots; one aliquot is centrifuged to obtain the plasma; the second aliquot is sonicated to disrupted the cells; both aliquots are centrifuged and treated with precipitating agent.
Which is the correct method?
2. How calculate I the absolute value of my compound in plasma and in cellular fraction? Do I have need of haematocrit?
Thank you for your answers.
With my regards.
Section of Experimental and Clinical Pharmacology
Departement of Clinical Medicine
University of Insubria
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The following message was posted to: PharmPK
I am assuming that your drug is stable in whole blood. Blood is rich in
esterases that may chop your drug if it is a substrate for it and you may
not be able to get a correct mass balance. To establish that you could
spike your drug in an equal aliquot of plasma and whole blood and monitor
for drug disappearance. If plasma samples do not show a fall in the spiked
initial drug concentration then probably your drug is not a substrate, but
if your drug is disappearing in the whole blood then it may be permeating
into the cells which you could investigate the steps below for drug in
First to measure the drug in plasma and cells you should have a method that
is validated for each matrix used. To determine the partitioning of your
drug into the cellular fraction you should first spike the drug in whole
blood and make two aliquots. In the first aliquot incubate at 37*C for a
predetermined time period and then centrifuge the spiked sample to obtain
the blood cells. Then you could re-suspend the sediment in PBS or other
appropriate buffer (that does not rupture the cells) to get rid of the
plasma that may have trace amounts of your drug in it, then centrifuge and
remove the supernatant. Repeat this step 3-4 times. Then you could sonicate
the cells and further analyze the sample for permeated drug. The second
aliquot you could centrifuge and measure the plasma. There are several
papers that have been published over the years. Care must be taken to
ensure that whole blood is not hemolyzed. It is good to have an estimate of
hematocrit in hand that could be used in your calculations depending upon
Hope this helps.
Manish Issar, Ph.D
Applied Biopharmaceutics, LLC
2010 Cascade Drive
Corona, CA 92879
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Collect the blood. Separate the plasma (call it as a reference plasma) from the part of that blood. Now incubate the same concentration of compound both in blood and plasma. At the end of incubation, separate the plasma from compound incubated blood (call it as test plasma). Compare the concentrations or apply some formula incorporating hematocrit in both reference plasma and test plasma.
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